This invention relates to the use of an indole-3-acetic acid (IAA) analogue as a plant hormone stimulatory to plant growth, to regeneration of plant cells and tissues, and to transformation of plant cells. It particularly relates to the use of mono- and multi-substituted IAA molecules. The invention also relates to compositions comprising IAA analogues of the invention.
Plant growth is affected by a variety of physical and chemical factors. Physical factors include available light, day length, moisture and temperature. Chemical factors include minerals, nitrates, cofactors, nutrient substances and plant growth regulators or hormones, for example, auxins, cytokinins and gibberellins.
Indole-3-acetic acid (IAA) is a naturally-occurring plant growth hormone identified in plants. IAA has been shown to be directly responsible for the increase in growth in plants in vivo and in vitro. The characteristics influenced by IAA include cell elongation, internodal distance (height), leaf surface area and crop yield. IAA and other compounds exhibiting hormonal regulatory activity similar to that of IAA are included in a class of plant regulators called xe2x80x9cauxins.xe2x80x9d
Preparations based on cytokinins, such as 6-furfurylamino purine (kinetin) and 6-benzylamino purine (BAP), are also known to be growth stimulators. However, cytokinin-based preparations are most effective in combination with auxins. While the mechanism by which cytokinins affect the growth cycle of plants is far from being understood, it is apparent that they affect leaf growth and prevent aging in certain plants.
It is a general objective in the field to successfully engineer and regenerate plants of major crop varieties using methods such as tissue culture and genetic engineering. Major crop varieties of particular interest in this regard are agricultural crops such as maize, wheat, rice, soybeans and cotton.
To regenerate plants, in vitro culture techniques have been established. (Reinert, J., and Bajaj, Y. P. S., eds. (1977) Plant Cell, Tissue and Organ Culture, Berlin: Springer; Simmonds, N. W. (1979) Principles of Crop Improvement, London: Longman; Vasil, I. K., Ahuja, M. K. and Vasil, V. (1979) xe2x80x9cPlant tissue cultures in genetics and plant breeding,xe2x80x9d Adv. Genet. 20:127-215.) Specific in vitro culture techniques to regenerate plants include embryo culturing shoot tip culturing and callus, cell and protoplast culturing. Embryo culturing has been shown to be important in making difficult interspecies crosses, while shoot-tip culturing is important in rapid clonal multiplication, development of virus-free clones and genetic resource conservation work. Callus, cell, and protoplast cultures have been shown to be important for cultures in which organization is lost but can be recovered.
Plant genetic engineering techniques have also been established. These techniques include gene transfer by transformation or by protoplast fusion. In gene transfer, the steps involved are: (a) identification of a specific gene; (b) isolation and cloning of the gene; (c) transfer of the gene to recipient plant host cells: (d) integration, transcription and translation of the DNA in the recipient cells; and (e) multiplication and use of the transgenic plant (T. Kosuge, C. P. Meredith and A. Hollaender, eds (1983) Genetic Engineering of Plants, 26:5-25; Rogers et al. (1988) Methods for Plant Molecular Biology Academic Press, Inc., San Diego, Calif.). In protoplast fusion, plant cell protoplasts are fused by standard chemical (e.g., PEG) or electroporation techniques. After regeneration of the fused cells, interspecies amphidiploids have been obtained. The technique may provide desired amphidiploids which cannot be made by conventional means, and presents possibilities for somatic recombination by some variant of it. The foregoing techniques are widely in use (Chaleff, R. S. (1981) Genetics of Higher Plants, Applications of Cell Culture, Cambridge: Cambridge University Press), and newly inserted foreign genes have been shown to be stably maintained during plant regeneration and are transmitted to progeny as typical Mendelian traits (Horsch et al. (1984) Science 223:496, and DeBlock et al. (1984) EMBO 3:1681). These foreign genes retain their normal tissue specific and developmental expression patterns.
The Agrobacterium tumefaciens-mediated transformation system has proved to be efficient for many dicotyledonous plant species. For example, Barton et al. (1983, Cell 32:1033) reported the transformation and regeneration of tobacco plants, and Chang et al. (1994, Planta 5:551-558) described stable genetic transformation of Arabidopsis thaliana. 
The Agrobacterium method for gene transfer was also applied to monocotyledonous plants, e.g., in plants in the Liliaceae and Amaryllidaceae families (Hooykaas-Van Slogteren et al., 1984, Nature 311:763-764) and in Dioscorea bulbifera (yam) (Schafer et al., 1987, Nature 327:529-532); however, this method did not appear to be efficient for the transformation of graminaceous monocots, which include such food crops as wheat, rice and corn.
Transformation of food crops was obtained with alternative methods, e.g., by polyethylene glycol (PEG)-facilitated DNA uptake (Uchimiya et al. (1986) Mol. Gen. Genet. 204:204-207) and electroporation (Fromm et al. (1986) Nature 319:791-793), both of which used protoplasts as transfer targets. Monocot and dicot tissues may be transformed by bombardment of tissues by DNA-coated particles (Wang et al. (1988) Plant Mol. Biol. 11:433-439; Wu, in Plant Biotechnology (1989), Kung and Arntzen, Eds., Butterworth Publishers, Stoneham, Mass.). Regeneration was described in rice (Abdullah et al. (1986) Bio/Technology 4:1087-1090) and maize (Rhodes et al. (1988) Bio/Technology 6:56-60 and (1988) Science 240:204-207).
Thus, although regeneration and transformation protocols have been established, there remains a need to stimulate regeneration and transformation of monocotyledonous and dicotyledonous plants. Indeed, some plants have been difficult to regenerate and transform [Vasil and Vasil (1994) in Plant Cell and Tissue Culture (Vasil and Thorpe, eds.), Kluwer Academic Publishers, Dordrech, Netherlands; Chee (1995) Plant Cell Reports 14:753-757; Bums and Schwartz (1996) Plant Cell Reports 15:405-408; Mihaljevic et al. (1996) Plant Cell Reports 15:610-614; Schopke et al. (1996) Nature Biotechnology 14:731). Moreover, there is a need to stimulate growth of the plants, particularly after transformation and regeneration.
The present invention satisfies these needs by providing compounds and compositions which stimulate plant growth, regeneration of plant cells and tissues, and transformation of plant cells and tissues. The compounds of the invention comprise mono- or multi-substituted IAA (indole-3-acetic acid) or ester or salt derivatives thereof. The invention also provides compositions comprising one or more of these IAA analogues and, optionally, a carrier. The invention contemplates the use of such auxinic analogues to affect growth, regeneration and transformation in both monocotyledonous and dicotyledonous plants. In particular, the invention provides monosubstituted IAA analogues having a substituent group at the 2, 4, 5, 6 or 7 position of the IAA structure, wherein said substituents are halo- or alkyl-, alkoxy-, acyl-, acylamido- and acyloxy-substituent groups having 1-10 carbon atoms. The invention also provides multi-substituted IAA analogues having two to five, same or different, substituent groups at different positions selected from positions 2, 4, 5, 6 or 7 of the IAA structure wherein said substituents are halo- or alkyl-, alkoxy-, acyl-, acylamido- and acyloxy-substituent groups having 1-10 carbon atoms.
The compositions of the invention may include, in addition to one or more of the mono- or multi-substituted compounds, one or more additional plant growth regulators. Such plant growth regulators include, for example, a cytokinin, a gibberellin, etc., in definite proportions for wide application to various plants. In specific embodiments, the invention is exemplified with compositions comprising mono- or multi-substituted IAA analogues having between one and five, same or different, substituent groups that are halo-, alkyl-, alkoxy-, acyl-, acylamido- or acyloxy-substituent groups at positions 2, 4, 5, 6 and/or 7 of the IAA structure, and a cytokinin to affect the growth of plants.
The invention further relates to media comprising the compounds and compositions of the invention. Such media comprise one or more IAA analogues and optionally an IAA analogue and optionally a plant growth regulator, e.g., cytokinin, to stimulate plant growth, to stimulate regeneration of plant cells and tissues, and to stimulate transformation of plant cells and tissues.
The invention also relates to a method of stimulating plant growth comprising (a) applying to a plant, plant cell or tissue an effective amount of the compound or composition of the invention and optionally, applying one or more additional plant growth regulators, for example, a cytokinin, a gibberellin, etc., and (b) incubating the plant cell or tissue under conditions sufficient to stimulate the regeneration of the plant cell or tissue.
The invention also provides a method for stimulating the regeneration of plant cells and/or tissues comprising (a) applying to a plant cell or tissue an effective amount of the compound or composition of the invention and applying one or more additional plant growth regulators, for example, a cytokinin, a gibberellin, etc., and (b) incubating the plant cell or tissue under conditions sufficient to stimulate the regeneration of the plant cell or tissue.
Also, the invention provides a method for stimulating the transformation of plant cells and/or tissues comprising (a) contacting the plant cell or tissue with a nucleic acid molecule (e.g., by transformation or protoplast fusion), (b) applying to the plant cell or tissue an effective amount of a compound or composition of the invention and, optionally, one or more additional plant growth regulators, for example, a cytokinin, a gibberellin, etc., and (c) incubating the plant cell or tissue under conditions sufficient to stimulate transformation of the plant cell or tissue with the nucleic acid molecule. The compounds and compositions of the invention also may be used to stimulate regeneration or growth of the transformed tissue or cells, thus providing a method to obtain a transgenic plant.
The invention also concerns a method of attenutating or alleviating environmental stress in a plant, plant cell or tissue comprising (a) contacting a plant, plant cell or tissue which has been exposed to an environmental stress (such as drought, excess temperature, diminished temperature, chemical toxicity [e.g., antibiotic, herbicides], pollution, excess light, and diminished light) with an effective amount of the compounds or compositions of the invention, and (b) incubating said plant, plant cell or tissue under conditions sufficient to attenuate or alleviate said stress.